Human babesiosis, a malaria-like febrile illness is an emerging tick-borne disease caused by Babesia microti (Bm), which is maintained in a similar enzootic cycle as Borrelia burgdorferi, the Lyme disease agent. Bm is the most common transfusion-transmitted pathogen in the United States and results in a ~20% mortality rate in transfusion recipients and other immunocompromised hosts. A critical bottleneck for epidemiological and evolutionary studies of this and other vector-borne pathogens has been the difficulty in obtaining sufficient numbers of whole genome sequences (WGS) of pathogens distributed across their geographic and host distribution. The only means to capture the complete diversity spectrum of vector-borne pathogens is to sequence directly from Ixodes scapularis nymphal ticks, because nymphs feed as larvae on all potential reservoir hosts. However, because of the small genomic size of the pathogen relative to the host, and their often low copy number in mixed DNA samples, the pathogen's genome signal is swamped by exogenous DNA, rendering next generation shotgun sequencing for these templates inefficient and costly. A novel approach is required to provide the epidemiological and clinical communities with genomic resources for a variety of downstream applications. We propose a novel culture-independent method for deriving whole genome sequences for vector-borne and zoonotic pathogens, allowing pathogen genomic variation to be studied directly from tick and human blood samples and thus enabling analyses at an unprecedented resolution. Specifically, we will adapt DNA target capture techniques, previously used in human genomic analyses, to probe the genomic diversity of B. microti in 120 strains sampled directly from field collected I. scapularis ticks, as well as 20 strains from human blood. This approach has never before been applied to vector-borne and zoonotic pathogens. Using these genomic resources, we will analyze the patterns of standing B. microti genetic diversity and characterize patterns of B. microti spread in the northeastern United States. This exploratory analysis will elucidate the degree of B. microti spatial population structure, identify the origin of human infective strains, and enable us to reconstruct B. microti invasion history across the Northeast. Together with understanding of the pathogen population structure, we will define the pool of parasites that can give rise to human disease, thereby contributing to future disease surveillance and control strategies. These baseline data will enable future epidemiological studies and clinical investigations aimed at understanding the mechanisms underlying human infectivity and provide the basis for parasite diagnostics relative to lineage-specific variation in important traits such as infectivity and disease severity.